UNIVERSITY OF KWAZULU NATAL
THE EFFECTS OF Sutherlandia frutescens IN CULTURED RENAL
PROXIMAL AND DISTAL TUBULE EPITHELIAL CELLS
BY
ALISA PHULUKDAREE
B.Sc., B.Med.Sc. (Hons), (UKZN)
Submitted in partial fulfilment of the requirements for the degree of M.Med.Sci
in the
Discipline of Medical Biochemistry,
Faculty of Health Sciences
University of KwaZulu-Natal
2009
ABSTRACT
Sutherlandia frutescens (SF), an indigenous medicinal plant to South Africa (SA), is traditionally used to treat a diverse range of illnesses including cancer and viral infections. The biologically active compounds of SF are polar, thus renal elimination increases susceptibility to toxicity. This study investigated the antioxidant potential, lipid peroxidation, mitochondrial membrane potential and apoptotic induction by SF on proximal and distal tubule epithelial cells. Cell viability was determined using the MTT assay. Mitochondrial membrane potential was determined using a flow cytometric JC-
1 Mitoscreen assay. Cellular glutathione and apoptosis were measured using the GSH-GloTM
Glutathione assay and Caspase-Glo® 3/7 assay, respectively. The IC50 values from the cell viability results for LLC-PK1 and MDBK was 15 mg/ml and 7 mg/ml, respectively. SF significantly decreased intracellular GSH in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. Lipid peroxidation increased in LLC-PK1 (p < 0.0001) and MDBK (p < 0.0001) cells. JC-1 analysis showed that SF promoted mitochondrial membrane depolarization in both LLC-PK1 and MDBK cells up to 80% (p <
0.0001). The activity of caspase 3/7 increased both LLC-PK1 (11.9-fold; p < 0.0001) and MDBK (2.2- fold; p < 0.0001) cells. SF at high concentrations plays a role in increased oxidative stress, altered mitochondrial membrane integrity and promoting apoptosis in renal tubule epithelia.
i DECLARATION
This study represents the original work by the author and has not been submitted in any form to another University. The use of work by others has been duly acknowledged in the text.
The research described in this study was carried out in the Discipline of Medical Biochemistry, Faculty of Health Sciences, University of KwaZulu-Natal, Durban, under the supervision of Prof. A. A.
Chuturgoon.
______
Alisa Phulukdaree
ii ACKNOWLEDGEMENTS
I would like to thank:
• Prof. A.A. Chuturgoon, for your encouragement, guidance and constructive criticism. Your
success as a researcher and unwavering spirituality is truly inspiring.
• My Family, for their support, love and patience during my undertaking of this research. To my
Mum, Anetha Phulukdaree, I am truly grateful for the sacrifices that you have made for me in these
recent years to help me achieve my ambitions. The spirit of my Dad guiding and protecting me, I
pray that you are pleased.
• UKZN’s LEAP Mellon Foundation for the scholarship that provided financial assistance which enabled completion of this degree.
• Dr. Devapregasan Moodley for your friendship, support and technical guidance.
• All academic staff and senior postgraduate students of the Mycotoxin Research Laboratory for their encouragement and support.
iii ABBREVIATIONS
∆Ψm Mitochondrial membrane potential
AIDS Acquired immunodeficiency syndrome
AIF Apoptosis inducing factor
ALA Alpha lipoic acid
Apaf-1 Apoptotic protease activating factor-1
ATM Ataxia telangiectasia mutated protein
ATP Adenosine triphosphate
ATR ATM related
Bcl-2 B cell lymphoma-2
. CCl3 Trichloromethyl
CCM Complete culture media
CD4+ Cluster differentiation
CHO Chinese hamster ovary
Complex IV Cytochrome oxidase
Complex V Mitochondrial ATP synthase
COX Cyclooxygenase
CYP Cytochrome P450 enzymes
DCT Distal convoluted tubule
DED Death effector domain
DHLA Dihydrolipoic acid
DISC Death inducing signalling complex
DMSO Dimethyl sulphoxide
DNA Deoxyribose nucleic acid
iv DNA-PK DNA-dependant protein kinase
ETC Electron transport chain
FADH2 Flavin-adenine dinucleotide dihydrogen
FADD Fas associated death domain
FMN Flavin mononucleotide
GABA Gamma-amino-butyric-acid
GABA-T GABA transaminase
GAD Glutamate decarboxylate
GSH Glutathione
GSSG Oxidised glutathione h hour/s
H2O2 Hydrogen peroxide
HIV Human immunodeficiency virus
HL60 Promyelocyte cell line
IAP’s Inhibitors of apoptosis proteins
IL-1β Interleukin one beta
IC50 Concentration of 50% cell growth inhibition i.p intraperitoneal
JNK Jun NH2-terminal kinase
MDA Malondialdehyde
Mdm2 Mouse double minute 2
NADH reduced nicotinamide adenine dinucleotide
NADPH reduced nicotinamide adenine dinucleotide phosphate
NF-κB Nuclear Factor kappa B
NO Nitric oxide v NOS Nitric oxide synthase
O2 Oxygen
.- O2 Superoxide
OH. Hydroxyl radical
OONO- Peroxynitrite p53 Tumour suppressor protein p53
PCT Proximal convoluted tubule
PG Prostaglandin
PUFA Polyunsaturated fatty acids
RIP Receptor-interacting protein
ROS Reactive oxygen species
RS. Thiyl
RT Room temperature
SA South Africa s.e. Standard deviation
Smac Second mitochondrial activator of caspases
SOD Superoxide dismutase
STZ streptozotocin tBid truncated bid
TNF-α Tumour necrosis factor alpha
TNFR1 Tumour necrosis factor receptor 1
TRADD TNFR1-associated death domain
TRAF2 TNF-associated factor 2
TRAIL Tumour necrosis factor related apoptosis inducing ligand vi LIST OF FIGURES
Chapter 1 Legend Page
Figure 1.1 Chemical structure of pinitol…………………………………………………….…6
Figure 1.2 The relationship of GABA to pathways in metabolism…………………………….7
Figure 1.3 The transmembrane proteins of the inner mitochondrial matrix
involved in the electron transport chain and oxidative phosphorylation………..….14
Figure 1.4 Structure of glutathione……………………….……………………………………17
Figure 1.5 Programmed cell death – extrinsic and intrinsic activation (Hengartner, 2000)…...20
Chapter 2 Legend Page
Figure 1 Levels of GSH in LLC-PK1 and MDBK cells treated with SF………...... (SAJS, Vol 106) 56
Figure 2 Levels of MDA in SF treated LLC-PK1 and MDBK cells…………….....(SAJS, Vol 106) 56
Figure 3 The ∆Ψm in LLC-PK1 and MDBK cells treated with SF after 48h………(SAJS, Vol 106) 57
vii LIST OF TABLES
Chapter 2 Legend Page
Table 1 Cell viability of PCT and DCT cells incubated with SF for 48h…..…..(SAJS, Vol 106) 55
Table 2 Caspase activity of PCT and DCT cells incubated with SF for 48h…..(SAJS, Vol 106) 57
viii TABLE OF CONTENTS
ABSTRACT………………………………………………………………………………………….....i
DECLARATION………………………………………………………………….……………………ii
ACKNOWLEDGEMENTS…………………………………………………………………………...iii
ABBREVIATIONS……………………………………………………………………………………iv
LIST OF FIGURES…………………………………………………………………………………..vii
LIST OF TABLES…………………………………………………………………………………...viii
TABLE OF CONTENTS……………………………………………………………………………...ix
INTRODUCTION……………………………………………………………………………………...1
CHAPTER 1: LITERATURE REVIEW……………………………………………………………..3
1.1 Sutherlandia frutescens …………………………………………………………………………….3
1.1.1 Adaptogenic properties, Uses and Distribution of Sutherlandia frutescens…………………..3
1.1.2 Toxicity………………………………………………………………………………………...4
1.1.3 Description and Biologically Significant Components of Sutherlandia frutescens…………4
1.1.4 L-Canavanine…………………………………………………………………………………..5
1.1.5 Pinitol…………………………………………………………………………………………..6
1.1.6 Gamma amino butyric acid…………………………………………………………………….6
1.1.7 Sutherlandia frutescens tablets…………………………………………………………………7
1.1.8 Pharmacology of Sutherlandia frutescens……………………………………………………...8
1.1.8.1 Stress…………………………………………………………………………………8
1.1.8.2 Epilepsy………………………………………………………………………………8
1.1.8.3 Antioxidant properties………………………………………………………………..8
ix 1.1.8.4 Anti-inflammatory properties………………………………………………………...9
1.1.8.5 Antiviral activity……………………………………………………………………..9
1.1.8.6 Diabetes………………………………………………………………………………9
1.1.8.7 Apoptotic properties of Sutherlandia frutescens……………………………………10
1.2 The Nephron………………………………………………………………………………………..12
1.2.1 Function of the Nephron……………………………………………………………………...11
1.2.2 Structure of the Nephron……………………………………………………………………...11
1.3 Biochemical Activity of PCT and DCT Epithelial Cells…………………………………………..12
1.3.1 Energy Production…………………………………………………………………………….12
1.3.2 Free Radicals………………………………………………………………………………….15
1.3.3 Antioxidants…………………………………………………………………………………..16
1.3.4 Cells Response to Stress………………………………………………………………………18
1.4 The Fate of the Cell………………………………………………………………………………...18
1.4.1 Apoptosis……………………………………………………………………………………...19
1.4.1.1 Extrinsic Apoptotic Activation……………………………………………………...19
1.4.1.2 Intrinsic pathway of activation for apoptosis……………………………………….21
1.4.1.3 Caspases – Effectors of Apoptosis………………………………………………….22
CHAPTER 2: SCIENTIFIC PAPER PUBLICATION…………………………………………….24
CHAPTER 3: CONCLUSION……………………………………………………………………….30
REFERENCES………………………………………………………………………………………..31
APPENDIX 1………………………………………………………………………………………….39
x INTRODUCTION
Customarily, toxicology has been termed “the science of poisons” (Langman and Kapur, 2006). This science engages studying the properties of chemicals and the impact of these molecules on living organisms. Toxicological studies have provided a tool which considers the potential undesirable effects of chemicals in order to maintain and protect human health (Roberfroid, 1995).
Historically, toxicology dates as far back as 300 B.C where early man used animal venoms and plant extracts for hunting and as “bio-weapons” during war. Plant extracts formed the basis of therapeutics and experimental medicine during the early centuries. The discipline of toxicology integrates the understanding and applications of the biological sciences, chemistry, physics and mathematics to test its theories. Toxicology differs from other sciences with the absence of a single goal but its diversification has allowed for the interspersion of ideas and concepts from academe, industry as well as government. This gives the discipline of toxicology a unique but highly advantageous slant (Amdur et al., 1991).
The majority of Africa’s population reside in rural areas where there is a lack of basic health care facilities. These people are thus reliant on traditional remedies to treat a range of diseases. Traditional healers make use of herbal medicines despite the lack of evidence for its safety. It is therefore imperative that the identification, efficacy, therapeutic doses, toxicity, standardisation and regulation of these plants and their extracts be determined (Chattopadhyay, 2003).
The popular multi-purpose herbal remedy used by South Africans is Sutherlandia frutescens (SF).
Sutherlandia frutescens is believed to have therapeutic potential against viral diseases, cancer, diabetes and a range of other conditions (Fernandes et al., 2004). Herbal remedies are currently receiving much
1 attention from the general public so much so, that these remedies are being made available in urban areas in the form of tablets. The spotlight, however, has shifted from the traditional conclusions of efficacy to the laboratories where experimentation will either prove or disprove the therapeutic potential to provide a cost effective and natural solution to health ailments.
2 CHAPTER 1
LITERATURE REVIEW
1.1 Sutherlandia frutescens
Traditional medicine is a common resort of majority of Africans that require healthcare as it is difficult and unaffordable to access healthcare facilities (Ojewole, 2008). In 2005 Mills et al. reported that approximately half of all pharmaceuticals were derived from plants. The diverse array of medicinal plants found in South Africa is estimated to be approximately 3 000 and is used regularly by the rural populace (van Wyk, 2008). A popular medicinal plant which is now commercially available due to its multi-purpose uses is SF. This plant has been used for a long time and it is assumed to be safe (Mills et al., 2005). The chemistry, pharmacology and toxicity of SF extracts are currently under critical analysis due to the increased usage and assumed safety of SF.
1.1.1 Adaptogenic properties, Uses and Distribution of Sutherlandia frutescens
Sutherlandia frutescens also referred to as the ‘cancer bush’ has been used for years by traditional healers to treat a variety of ailments. These include internal cancers, diabetes, uterine disease, influenza, human immunodeficiency virus (HIV) infection, depression, and arthritis (Gericke et al.,
2001).
Sutherlandia frutescens belongs to the family: Fabraceae/Leguminosa and is one of five currently recognised Sutherlandia species, all of which are indigenous to South Africa. This plant is distributed mainly in the Western Cape and Karoo regions (Fernandes et al., 2004) in South Africa.
3 1.1.2 Toxicity
The recommended therapeutic dose of SF leaf powder for humans is 9mg/kg body weight/day with no side effects (Sia, 2004). This was determined during a toxicity study conducted on vervet monkeys
(Medical Research Council and National Research Foundation of South Africa, 2002). During this study the dosage was increased nine times with no resulting haematological, clinical and physiological toxicity (Mills et al., 2005).
A clinical trial conducted on healthy human volunteers showed no significant adverse changes in parameters following a daily dose of 800mg for 3 months (Ojewole, 2008). The commercially available SF tablet has a recommended dosage of 600mg/day.
1.1.3 Description and Biologically Significant Components of Sutherlandia frutescens
Sutherlandia frutescens is a perennial short-lived shrub that grows between 0.2-2.5m in height and has petiolate, stipulate and pinnate leaflets of 8-10 pairs with a terminal leaflet. Red and, seldomly white, flowers bear in axillary racemes and produces large and bladder-like pods containing numerous brown, laterally compressed, kidney shaped seeds. Leaves of SF are mainly used traditionally but all aerial parts are thought to have medicinal properties.
Biologically active compounds that have been isolated from SF include pinitol, flavonoids, saponins and amino acids such as gamma-amino-butyric-acid (GABA), L-canavanine (van Wyk, 2008), arginine, asparagine, proline, alanine, leucine, tryptophan and phenylalanine (Tai et al., 2004). Other components of SF, along with commonly found plant-derived alcohols, include hexadecanoic acid, propyl parabens, methyl parabens, gamma sitosterol and sigmast-4-en-3-one (Sai, 2004).
4 1.1.4 L-Canavanine
L-canavanine is found at approximately 14.5mg per gram of dry SF leaves (van Wyk and Albrecht,
2008) and 3mg per gram in Phytonova SU1 SF tablets. Most leguminous plants in the subdivision
Leguminosae synthesise L-canavanine (Bence and Crooks, 2003) which is a storage form of nitrogen in plant seeds and is used in the chemical defence mechanisms (Rosenthal, 1977).
L-canavanine is the L-2 amino-4-guanidinooxy structural analogue of L-arginine. The electron density of the guanidino group is reduced due to the destabilising effect of the oxygen atom (Bence and
Crooks, 2003). As a result, the guanidino group of L-arginine has a pKa of 12.48 compared to 7.05 for the oxyguanidino group of L-canavanine (Swaffar et al., 1994). This makes L-canavanine less basic than L-arginine and exists in the amino, instead of the imino tautomeric form. These simple differences have huge implications with regard to their physiological roles. L-arginine is an important amino acid that is necessary for the normal development and growth of cells (Bence and Crooks, 2003) while L-canavanine is cytotoxic (Swaffar et al., 1994).
L-canavanine has been shown to be a potent inhibitor of inducible nitric oxide synthase (NOS)
(Lincoln et al., 1997), as well as other arginine-utilising enzymes (Hrabak et al., 1994). Nitric oxide synthase utilises L-arginine for the biosynthesis of nitric oxide (NO) and L-citrulline. The NOS reaction occurs in two steps, each using molecular oxygen (O2) and reduced NADPH as co-substrates
(Luzzi and Marletta, 2005). L-canavanine, a substrate in the NOS reaction, is converted to L- homoserine by the enzyme.
L-canavanine is responsible for structural changes induced in proteins when it is incorporated into a growing peptide chain in place of L-arginine. The outcome of the replacement is the initiation of
5 functional changes in the proteins containing L-canavanine. Thus the anti-metabolic properties of L- canavanine can be explained.
1.1.5 D-Pinitol
Figure 1.1 Chemical structure of D-pinitol (Do et al., 2008).
D-pinitol (Figure 1.1) is a legumous chiro-inositol sugar found at levels of 14mg per gram of dry SF leaf (Moshe, 1998; van Wyk and Albrecht, 2008). D-pinitol possesses anti-diabetic properties (Bates et al., 2000) and has been implicated in treating wasting in cancer and acquired immunodeficiency syndrome (AIDS) (Ostlund and Sherman, 1996). This insulin-like effect makes glucose readily available to cells for increased metabolism, thus increasing intracellular ATP levels. Pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α) and interleukin one beta (IL-1β) was reduced in rats with acute oedema treated with D-pinitol (Sia, 2004).
1.1.6 Gamma amino butyric acid
In SF tablets there is more or less 0.4mg/g of GABA (Tai et al., 2004). Gamma amino butyric acid is an inhibitory neurotransmitter which mediates its effects outside the nervous system (van Wyk, 2008).
This amino acid has been used and is thought to be responsible for relief of anxiety and stress (Sia,
2004).
6
Figure 1.2 The relationship of GABA to pathways in metabolism
(Shelp et al., 1999).
In general, GABA was found to affect the absorption of ions in the renal tubules (Parducz et al., 1992).
Intracellularly, GABA is shunted along enzymes, glutamate decarboxylate (GAD), GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase to form intermediates that can enter the Kreb’s cycle (Figure 1.2).
1.1.7 Sutherlandia frutescens tablets
Phyto Nova SutherlandiaTM Tablets are readily available as an over the counter complementary medicine. It is suggested that one tablet be taken twice a day after meals. Each tablet contains 300mg milled select SF subspecies microphylla chemotype. Side effects that have been documented include observed dizziness, loose stool, mild constipation and dry mouth (Mills et al., 2005).
7 1.1.8 Pharmacology of Sutherlandia frutescens
1.1.8.1 Stress
Stress is often associated with increases in circulating levels of steroid hormone. Sutherlandia frutescens is believed to have stress alleviating properties. This facet of SF was elucidated in a study by Prevoo et al. (2008) where experimental results indicated the attenuation of adrenal cytochrome
P450 enzymes, CYP17 and CYP21. These enzymes are actively involved in the biosynthesis of steroid hormones. The potential for SF to depress the activity of these enzymes provide insights to the mechanism behind the stress-relieving effects observed (Prevoo et al., 2008).
1.1.8.2 Epilepsy
Many experiments are currently being conducted to determine the mechanism by which SF exerts its biological effect/s in cells. In an in vivo experiment, SF (50-400 mg/kg i.p) was found to possess significant anticonvulsant and antiseizure effects in Balb/C mice following epilepsy induction by pentylenetetrazole (Ojewole, 2008).
1.1.8.3 Antioxidant properties
Luminal and lucigenin enhanced chemiluminescence was used to investigate the hydroxyl free radical and superoxide scavenging properties of an aqueous extract of SF. This study revealed that SF displayed anti-oxidant potential with concentrations as low as 10µg/ml in cell free and stimulated neutrophil systems (Fernandes et al. 2004).
In another study by Tai et al. (2004) the antioxidant capacity of a 70% ethanolic extract of SF was determined. This study showed that 0.5µl of the ethanolic SF extract was comparable with 10µM of
Trolox, an effective chemical with antioxidant activity.
8 1.1.8.5 Anti-inflammatory properties
During inflammation a vast array of factors are released and synthesised. One of these factors is the enzyme, cyclooxygenase (COX-2) which is involved in prostaglandin (PG) synthesis and inflammation. A study by Kundu et al. (2005) on the inflammatory properties of a methanolic extract of SF showed an inhibition of COX-2 induction in vitro and in vivo. In this study, tumour promoter 12-
O-tetradecanoylphorbol-13-acetate was applied on mice skin followed by SF. The expression of cyclooxygenase-2 was found to be suppressed by 26% and 48% in mice treated with 100mg and
200mg of the SF extract, respectively.
1.1.8.6 Antiviral activity
The resort for primary health care for many HIV+ individuals in South Africa is traditional herbal therapy as it is often difficult to access proper health care facilities (Mills et al., 2005). In 2005 Harnett et al. reported that HIV+ individuals who consumed SF showed an improvement in their mood, appetite, weight gain, cluster differentiation 4 positive (CD4+) counts as well as decreased viral loads.
It is thought that the antiretroviral activity of SF is through inhibition of HIV-1 target enzymes, such as
HIV-1 reverse transcriptase (Harnett et al., 2005).
1.1.8.7 Diabetes
A recent study showed that SF had hypoglycaemic properties and was useful in the treatment of type II diabetes. In this study crushed SF leaves in drinking water was administered to Wistar rats fed a high fat diet. The results showed that SF was an efficient hypoglycaemic mediator in comparison to metformin (the commonly used anti-diabetic drug) (Chadwick et al., 2007).
9 The hypoglycaemic effect of SF was also demonstrated in streptozotocin (STZ)-induced diabetic rats
(Type I diabetes) (Ojewole, 2004). This study found that SF caused significant hypoglycaemia in the
STZ-treated rats when dosed with concentrations of 50-800mg/kg i.p. of the extract.
1.1.8.8 Apoptotic properties of Sutherlandia frutescens
The SF plant is commonly referred to as the ‘cancer bush’ and is used by traditional healers to treat cancer. To determine whether SF possessed anti-cancer properties, human breast adenocarcinoma cells were exposed to an ethanolic extract of SF. The results showed morphological characteristics of apoptosis and cell growth inhibition by affecting the mitogen activated protein kinase pathway
(Stander et al., 2007, Stander et al., 2009).
The antiproliferative effect of SF was also demonstrated to be concentration dependant in breast cancer, the promyelocyte cell line HL60 and leukemia Jurkat cell line (Tai et al., 2004).
A separate study by Chinkwo (2005) demonstrated the apoptotic properties of SF on Caski cells
(cervical carcinoma), Chinese hamster ovary (CHO) and Jurkat T lymphoma cell line. The classical hallmarks of apoptosis such as cell shrinkage, breakdown and a decrease in cell numbers, was noted.
This study used the apoPercentageTM assay and Crossmon Trichrome stain which detects phosphatidylserine externalisation and chromatin condensation, respectively. Jurkat T cells treated with 3.5 mg/ml SF for 6 and 24 h were analysed by Annexin-V staining and flow cytometry showed approximately 84% apoptotic cells (Chinkwo, 2005).
Ethanolic extracts of SF was used to determine the apoptotic effects on the MCF-7 human breast carcinoma cell line (Stander et al., 2007). Microscopical analysis showed that SF treated cells displayed altered morphology. The changes included cytoplasmic shrinking, membrane blebbing and
10 apoptotic bodies - representing the classical characteristics of apoptosis. Changes in the expression of
345 genes in cells treated with SF were found using cDNA microarray analysis. Changes have been noted in genes which code for proteins involved in apoptosis, cell cycle regulation and signal transduction. These include TNF superfamily members 10a and 10b, caspase recruitment domain family, member11 amongst others (Stander et al., 2007).
1.2 The Nephron
1.2.1 Function of the Nephron
The route of elimination of polar compounds from the circulation occurs via the renal system. The kidney receives between 20% – 25% of the total cardiac output and therefore any substance in the systemic circulation reaches the kidney in large quantities. The functional unit of the kidney is the nephron. The kidney functions to filter blood, allowing substances to enter the Bowman’s capsule and renal tubules. Filtered nutrients are actively reabsorbed at the proximal convoluted tubule (PCT) and ions are actively reabsorbed at the distal convoluted tubule (DCT) (Klaassen, 2000). The proximal and distal tubules, based on their functions, have different cell architectures (Young et al., 2000).
1.2.2 Structure of the Nephron
The PCT epithelium (simple cuboidal epithelium) has a brush border of tall microvilli that extends into the lumen. This functions to increase the surface area 20-fold for the efficient reabsorption of molecules from the glomerular filtrate back into circulation.
Histologically, PCT cells stain intensely due to the high content of organelles and mitochondria. The
PCT is responsible for the active reabsorption of approximately 100% of glucose and amino acids from the glomerular filtrate (Young et al., 2000). The DCT cells are smaller simple cuboidal epithelial cells that stain less intensely due to the presence of fewer organelles. The DCT is involved in the active reabsorption of sodium from the tubular fluid and lacks a brush border (Young et al., 2000).
11 The close proximity of filtered substances to the renal tubular epithelium and the ability of the kidney to concentrate toxicants increase the susceptibility of these cells to damage.
1.3 Biochemical Activity of PCT and DCT Epithelial Cells
1.3.1 Energy Generation
The mitochondrion is considered the energy generator of all aerobic eukaryotic cells. This organelle is responsible for production of most of the energy, signalisation, biosynthesis and apoptotic mechanisms
(Saraste et al., 1999). The control of and the manner in which these cellular mechanisms occur plays a determinant role in cell physiology. The differences in energy utilisation lie not only between organs but are highly specific to the functionality of each cell type. Hence, the number and activity of organelles such as mitochondria in the proximal and distal convoluted tubular epithelial cells are different.
A distinctive feature of the mitochondria is the presence of a double membrane system, the inner and outer membranes. The outer membrane is not a significant permeability barrier as it contains transmembrane channel proteins (porins). The inner membrane is extremely convoluted forming cristae to increase the surface area and it presents a permeability barrier to most solutes. The inner mitochondrial membrane has transport proteins and protein complexes involved in electron transport and ATP synthesis. The high surface area provided by the cristae enhances the capacity of ATP production (Mathews et al., 2000).
Energy generation by the mitochondria occurs primarily through oxidative phosphorylation. This is a process by which electrons are passed along a series of carrier molecules in the electron transport chain
(ETC). These electrons are attained from the oxidation of reduced nicotinamide adenine dinucleotide
(NADH) and flavin-adenine dinucleotide dihydrogen (FADH2) which are produced during glycolysis,
12 Kreb’s cycle and other oxidation reactions during the metabolism of nutrients. Five enzyme complexes make up the ETC, complexes I, II, III and IV transport electrons and complex V catalyses the synthesis of ATP. These complexes function to accept electrons from electron carriers and transfer them to the next carrier in the chain. The electrons, ultimately, combine with protons and oxygen to produce water
(Mathews et al., 2000).
Complex I (NADH: ubiquinone oxidoreductase) receives the free proton from NADH and transfers it to flavin mononucleotide (FMN) producing reduced FMNH. Succinate: ubiquinone reductase (Figure
1.3) is also known as complex II which transfers electrons from succinate to flavin-adenine dinucleotide (FAD). Electrons from complexes I and II are then accepted by coenzyme Q which transports these electrons to complex III.
Complex III is responsible for the deliverance of electrons from coenzyme Q to cytochrome C in a redox reaction coupled by the generation of a proton gradient across the membrane. Cytochrome oxidase (complex IV) also generates a proton gradient against the transmembrane by receiving electrons from cytochrome c in its active site with haem iron and copper.
The active site of complex IV has a haem iron and a copper molecule that is uses to reduce oxygen into two water molecules utilising two protons from the mitochondrial matrix simultaneously pumping a proton across the membrane (Figure 1.3) (Mathews et al., 2000).
13
Figure 1.3 The transmembrane proteins of the inner mitochondrial matrix that are
involved in the electron transport chain and oxidative phosphorylation.
Complex I (NADH-DH), Complex II (SDH), Complex III (bc1), Complex IV
(COX) transfers electrons to oxygen. Complex I, III IV translocate protons
across the membrane creating a proton gradient that Complex V(ATP
synthase) uses to synthesise ATP (adapted from Mathews, 2000).
Mitochondrial ATP synthase (Complex V) synthesises ATP from ADP and Pi, using a proton motive force across the membrane and it can hydrolyse ATP to pump protons against an electrochemical gradient. It is made up of two major complexes (F1 and F0). The F0 complex is embedded within the inner membrane while the F1 complex protrudes into the matrix.
14 1.3.2 Free Radicals
Any molecule that is capable of independent existence when it contains one or more unpaired electrons
.- . is regarded as a ‘free radical’. This class of molecules include superoxide (O2 ), hydroxyl (OH ), thiyl
. . . (RS ), trichloromethyl (CCl3 ) and nitric oxide (NO ) (Halliwell and Chirico, 1993; Jones, 2006).
The oxygen-centred radicals are considered the most important in vivo generated free radicals. These reactive oxygen species (ROS) arise as a result of normal metabolic processes as well as physical irradiation. NADPH oxidase is responsible for the production of ROS in inflammatory cells during cell-mediated immunity and antimicrobial activity. The production of ROS by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in non-inflammatory cells is used for cell signalling. Superoxide generation in eukaryotic aerobic cells occurs in abundance at the mitochondrial
ETC during the synthesis of ATP (Cadenas and Sies, 1998).
Cytochrome oxidase, complex IV in the ETC, contains haem iron and copper which transfers one electron at a time to oxygen. This process is not efficient and therefore allows for the generation of incompletely reduced oxygen species (Jones, 2006). Depending on the number of electron reductions of oxygen different ROS are formed. A single electron reduction produces superoxide, two electron reductions produces hydrogen peroxide (H2O2) and three-electron reduction produces the hydroxyl radical (OH.) (Halliwell and Chirico, 1993).
The OH., most reactive of the ROS, has a half life of 10-9s and is responsible for the damage of biological molecules such as cellular lipids, proteins, and DNA (Pastor et al., 2000). Polynucleotide strand breakage, double stranded DNA breaks, base alterations and nucleotide base lesions occur as a
. result of OH damage (Mathews et al., 2000). Superoxide radicals and H2O2 may not be as detrimental
. . - as OH , however O2 readily reacts with NO to form peroxynitrite (OONO ) (Klatt & Lamas, 2000;
15 Ridnour et al., 2004). This reactive nitrogen species causes lipid peroxidation and protein damage by nitration of tyrosyl hydroxyl groups (Mathews et al., 2000).
The cell attempts to dissipate the superoxide radical by converting it to H2O2, a reaction catalysed by superoxide dismutase. Hydrogen peroxide, however, reacts with transition metals, iron and copper, during the Fenton reaction giving rise to highly reactive OH.. The OH. is then free to react with polyunsaturated fatty acids (PUFA) to produce a carbon-centred lipid radical (Halliwell and Chirico,
.- 1993). The lipid radical can then react with iron resulting in lipid alkoxyl radicals or with O2 forming lipid peroxyl radicals.
Lipid peroxidation is initiated when these reactive lipid peroxyl radicals are not detoxified by cellular antioxidants. The lipid alkoxyl radical undergoes cyclisation forming an intermediate product which can degrade into malondialdehyde (MDA). Nucleotide bases, cytosine, adenine and guanine, of DNA are targets of MDA which result in the formation of DNA adducts (Halliwell and Chirico, 1993).
1.3.3 Antioxidants
Oxidative stress is dealt with in vivo by both enzymatic and non-enzymatic antioxidant systems. The enzymatic antioxidant mechanisms include superoxide dismutase (SOD) and peroxidases. The non- enzymatic antioxidant system includes molecules such as vitamin C, vitamin E, beta-carotene, flavonoids, glutathione (GSH) and uric acid (Mathews et al., 2000).
.- Superoxide dismutase is a metalloenzyme that catalyses the dismutation of two O2 molecules to H2O2 and O2. Peroxidases include catalase and GSH peroxidase. Catalase reduces H2O2 to water and O2, and
GSH peroxidase reduces H2O2 to water along with the oxidation of GSH (Mathews et al., 2000).
16 Vitamin C (ascorbic acid) is a major extracellular antioxidant but is also capable of scavenging radicals in the aqueous phases of the cytoplasm. Vitamin E and beta-carotene act as antioxidants in hydrophobic environments (Mathews et al., 2000). Both vitamin C and vitamin E detoxify intermediates formed during lipid peroxidation. The lipid peroxyl radical is acted upon in the membrane by the reduced form of vitamin E forming lipid hydro-peroxide and a vitamin E radical.
Vitamin C then regenerates vitamin E by accepting the free radical and forming an ascorbyl radical.
The vitamin E radical can also be reduced by GSH. The oxidised GSH and ascorbyl radical are subsequently reduced to GSH and ascorbate monoanion by dihydrolipoic acid (DHLA) which is then converted to alpha-lipoic acid (ALA). Dihydrolipoic acid is regenerated from ALA using NADPH
(Mathews et al., 2000).
Glutathione (Figure 1.4), also called gamma-glutamylcysteinylglycine, is a tripeptide that is present in most cells (Sies, 1999). This molecule is responsible for protecting the cell against oxidative stress.
This is accomplished by acting as a cofactor for detoxification enzymes, scavenging hydroxyl radicals and singlet O2, detoxifying H2O2 and lipid peroxides. GSH also plays a role in the regeneration of vitamin C and vitamin E as mentioned previously.
Figure 1.4 Structure of glutathione (Sies, 1999).
17 An important feature of GSH is the presence of a free thiol group with which it forms conjugates with a range of electrophilic compounds nonenzymatically, or through the action of GSH–S-transferase
(Sies, 1999). The resulting GSH-conjugates are removed by the biliary system. They may also be metabolised further by hydrolysis and N-acetylation reactions resulting in mercapturic acid which is excreted by the renal system (Dickinson and Forman, 2002).
The reduction of peroxides by GSH peroxidase produces oxidised glutathione (GSSG). The depletion of GSH is circumvented by GSH reductase catalysing the reduction of GSSG to GSH using NADPH.
During oxidative stress the level of transcription of GSH peroxidase is up-regulated and the rate of post-translational modification is increased. Aberrant expression of this enzyme and its activity has been associated with many pathologies including hepatitis (Downey et al., 1998), HIV (Banki et al.,
1998) and cancers of various tissues including the kidney (Okamoto et al., 1994).
1.3.4 Cells Response to Stress
Cell signalling and signal transduction are the two means by which cells communicate with each other
(Poli et al., 2004). Extracellular signals such as hormones, cytokines, growth factors and neurotransmitters trigger signal transduction and allow information to be transferred from the outside to the intracellular elements (Thannickal and Fanburg, 2000). When a cell is placed under stress due to heat shock or xenobiotics, for example, several signalling cascades are activated to enhance cell repair mechanisms and promote cell survival.
1.4 The Fate of the Cell
Cells that are unable to recover from oxidative stress and chemical damage are signalled to undergo cell death. Cell death may occur either through necrosis or apoptosis. Necrosis usually occurs in cells following acute tissue injury and results in the release of intracellular molecules into the extracellular
18 matrix, initiating an inflammatory response. Tissue homeostasis and the demise of individually affected cells occur via programmed cell death/ apoptosis.
1.4.1 Apoptosis
A variety of mechanisms can induce apoptosis in cells. Intracellular pro- and anti-apoptotic factors, the severity of the stimulus and the stage of the cell cycle determine whether the signalled cell will undergo apoptosis or not. Apoptosis can be stimulated by extracellular or intracellular signals.
Extrinsic signals include binding of death inducing ligands (from the surface of other cells or soluble factors) to death receptors. Intrinsic apoptotic signals are produced following exposure to radiation, viral infection, growth factor deprivation or oxidative stress (Figure 1.5) (Hengartner, 2000).
1.4.1.1 Extrinsic Apoptotic Activation
Fas, tumour necrosis factor receptor 1 (TNFR1) and tumour necrosis factor related apoptosis inducing ligand (TRAIL), death receptor 4 and death receptor 5 are the best characterised and understood extrinsic death receptors which belong to the TNF-α superfamily.
The interaction of TNF-α with TNFR1 causes receptor trimerisation and the clustering of intracellular death domains (Van Antwerp et al., 1998). This allows intracellular TNFR1-associated death domain
(TRADD) to bind to the death domain followed by the recruitment of TNF-associated factor 2
(TRAF2) to the death receptor. The NF-kB and the Jun NH2-terminal kinase (JNK)/Ap-1 pathway are then activated by TRAF2.
19
Figure 1.5 Programmed cell death – extrinsic and intrinsic activation
(Hengartner, 2000).
The TNFR1 also recruits receptor-interacting protein (RIP)-associated ICH-1 homologous protein with death domain which uses caspase recruitment domain to recruit caspase 2. Fas associated death domain (FADD) interaction with TRADD also induces apoptosis by the recruitment and cleavage of procaspase 8 using its death effector domain (DED) (Ashe and Berry, 2003).
The death inducing signalling complex (DISC), formed by the interaction of procaspase 8 with the
DED of FADD, is a multi-protein complex and is responsible for the fate of the cell as its composition activates either apoptotic or survival pathways. The apoptotic pathway can occur rapidly by the recruitment of a high number of caspase 8 to DISC, or slowly with few caspase 8 molecules recruited and slow initiation of apoptosis following the involvement of the mitochondrial apoptotic pathway.
20 Caspase 8 cleaves downstream caspase 3 as well as pro-apoptotic molecule Bid to truncated Bid
(tBid). Second mitochondrial activator of caspases (Smac)/Diablo, apoptosis inducing factor (AIF), cytochrome c, and endonuclease G are mitochondrial factors whose release is stimulated by truncated bid (tBid) (Ashe and Berry, 2003).
1.4.1.2 Intrinsic pathway of activation for apoptosis
As described by Chowdhury et al. (2008), a range of factors including cell stress, oxidative stress, heat shock and DNA damage can lead to the intrinsic activation of apoptosis. The mitochondrion is an important organelle in the process of apoptosis as the mitochondrial membrane is the site at which anti-apoptotic proteins B cell CLL/lymphoma 2 (Bcl-2) and Bcl-X are located.
Pro-apoptotic molecules such as cytochrome c is located within the mitochondria and pro-apoptotic protein Bax and Bad mediate apoptosis by interacting directly with the mitochondrial membrane or with mitochondrial associates Bcl-2 and Bcl-X (Gross et al., 1998, Lalier et al., 2007). Bax is thought to have the ability of forming pores in the mitochondrial membrane thereby promoting the release of cytochrome C and AIF (Lalier et al., 2007). The release of cytochrome c, which may also be caused by cell stress, involved in apoptotic activation makes it available to form an apoptosome with apoptotic protease activating factor-1 (Apaf-1) and caspase 9 (Yang et al., 1997).
Damage to cellular DNA and protein leads to the generation of pro-apoptotic molecules such as Bax following the activation of p53 (Figure 1.5). The p53 protein, also known as the tumour suppressor protein, has been associated with key responses of the cell to DNA damage (Haffner and Oren, 1995).
The p53 protein plays an integral role in monitoring cellular stress and inducing apoptosis, when required (Hosfseth et al., 2004). The maintenance of appropriate levels of p53 is accomplished by mouse double minute 2 (Mdm2). This enzyme binds to the p53 protein and promotes its
21 polyubiquinitation and degradation, therefore maintaining low intracellular levels of this protein
(Brooks and Gu, 2003).
Changes in chromatin structure are a consequence of DNA damage. This phenomenon leads to activation of the chromatin-bound protein kinase, ataxia telangiectasia mutated protein (ATM), ATM related (ATR) and DNA-dependant protein kinase (DNA-PK) proteins which then phosphorylates p53
(Hickman et al., 2002; Kubbutat and Vousden, 1998). Activated p53 accumulates in the nucleus and binds to DNA sequences and induces transcription of proapoptotic proteins. p53 acts as a promoter of apoptosis by suppressing anti-apoptotic protein gene expression, Bcl-2, Bcl-xL (Yu and Zhang, 2005) and cFLIP (Ashe and Berry, 2003).
1.4.1.3 Caspases – Effectors of Apoptosis
The caspases, a group of cysteine proteases, are effectors of apoptosis that are present in cells in an inactive form. Stimuli that promote apoptosis initiate the cleavage of the procaspases thereby initiating the caspase cascade for the execution of apoptosis. The binding of appropriate ligands to death receptors activates the initiator caspases 10 and 8. These activate downstream caspases in the caspase cascade until the effector caspases 3, 6 and 7 are activated. The targets of effector caspases include nuclear lamins, ICAD/DNA fragmentation factor 45, PARP-1 and P 21- activated kinase 2 that result in morphological features of apoptotic cells (Kerr et al., 1972).
The intrinsic pathway of apoptosis ultimately results in the activation of executioner caspase 3 as well.
Release of cytochrome c from the mitochondria occurs in concert with Smac/DIABLO. The formation of the apoptosome with cytochrome c, apaf-1 and procaspase 9 renders caspase 9 active. The presence of inhibitors of apoptosis proteins (IAP’s) is able to react with caspase 9 and prevent further activation
22 of the caspase cascade. The release of Smac/DIABLO allows it to bind to IAP’s thereby allowing caspase 9 to cleave and activate caspase 3 (Ekert et al., 2001).
The features of apoptosis are DNA fragmentation by endogenous nucleases that are activated by effector caspases, cytoplasmic shrinkage due to the cleavage of lamins and actin filaments, chromatin condensation due to the degradation of nuclear structural proteins, externalisation of phosphatidylserine on the membrane of cells to promote phagocytosis, membrane blebbing and the formation of apoptotic bodies. This process is intricate and involves several protein families, sub cellular compartments and signal transduction cascades (Hengartner, 2000).
The availability, utilisation and popularity of medicinal plants are increasing amongst the South
African communities. Scientific evidence that supports the effectiveness of herbal therapies such as SF is emerging, proving its antiviral, antioxidant, antiproliferative and hypoglycaemic properties.
Naturally, traditional therapeutics is accepted by the populous; however, continued research by in vitro, in vivo (animal) experiments and clinical trials is imperative to validate the efficacy of SF.
23 CHAPTER 2
SCIENTIFIC PAPER PUBLICATION
PREFACE
The following paper has been published in the South African Journal of Science, Volume 106, Article
#10, Pages 54 - 58, documents the effect of Sutherlandia frutescens on cultured renal epithelial cells.
This study was undertaken in light of the lack of literature regarding the therapeutic value and
potential toxicity of the above medicinal plant.
24 25
26
27
28
29
CHAPTER 3
CONCLUSION
Sutherlandia frutescens is one of approximately 3 000 medicinal plants that is utilised daily to treat a wide range of illnesses in our population. These plants are now commercially available and their therapeutic potential remains to be elucidated. Continuous research into the safety and efficacy of the medicinal plant, SF, for human ailments need to be intensified.
The conclusions drawn from the current study indicate that SF is not cytotoxic to renal tubule epithelial cells at low concentrations. The plant extract at higher concentrations, however, induces cell death via apoptosis in renal tubule epithelia. Also, high concentrations of SF water extracts increased oxidative stress and altered the integrity of the mitochondrial membrane.
This in vitro experiment provides evidence that SF, if consumed moderately, may in essence; prove to be a safe therapeutic. Further research such as clinical trials are required to prove the safety and efficacy of this plant extract.
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38
APPENDIX 1
Graphical representation of cell viability (%) of cells exposed to SF for 48h.
39
40 Article #10 54 South African Journal of Science
1 close It has S Afr J Sci S 11 The used to Canaline, 5 21 Research Letter Research renal L-canavanine
2,3,4 Its production by 5,6 < 0.0001) and MDBK and 0.0001) < p 3/7 assay, respectively. assay, 3/7 ® An antiproliferative effect of
19 16,17 cultured
(11.9-fold; cells 1
in and MDBK cells by up to 80% 1
and MDBK were 15 mg/mL and 7 1
Based on their contrasting functions, the 20 9,10 Interestingly, GABA has been shown to affect 21 L-canavinine, a non-protein amino acid, is a
7 12 14 epithelial ts extrac
Intracellularly, GABA is metabolised through the action of ABSTRACT 13 Various doses of SF leaf powder have been administered to 1 INTRODUCTION The DCT cells are smaller, simple cuboidal epithelial cells (stain 21 < 0.0001) cells. JC-1 analysis showed that SF extracts promoted cycloartane glycosides and triterpenoid diglucoside. triterpenoid and glycosides cycloartane
p SF-treated human breast adenocarcinoma cells in culture showed tubule
15 < 0.0001) cells, while lipid peroxidation increased in treated LLC- Another study, by contrast, showed that SF extracts displayed antioxidant displayed extracts SF that showed contrast, by study, Another 2 p Commonly known as ‘cancer bush’, it has been used in crude form for years 1 (SF), a medicinal plant indigenous to South Africa, is traditionally cens frutes
(SF), a member of the Leguminosae family, is a multipurpose medicinal plant An apparent antiretroviral activity of SF has been thought to be mediated by the D-pinitol, a chiro-inositol sugar, possesses anti-diabetic properties and is used in 18 8 distal
Vol. 106 No. 1/2 Page 1 of 5 Vol. < 0.0001) cells. SF extracts at high concentrations appear to increase oxidative stress, to stress, oxidative increase to appear concentrations high at extracts SF cells. 0.0001) < p values from the cell viability results for LLC-PK SF contain the biologically active compounds L-canavanine, D-pinitol, gamma amino butyric 50
and < 0.0001) and MDBK (
p ( 1 < 0.0001) and MDBK ( LLC-PK both in increased 3/7 caspase of activity The 0.0001). < p p extracts alter mitochondrial membrane integrity, and to promote apoptosis in renal tubule epithelia. alter mitochondrial membrane integrity, and Sutherlandia frutescens treat a diverse range of illnesses, organ. that including in toxicity to susceptibility increases elimination cancer renal thus polar, are SF and of compounds viral infections. The biologically active This study investigated the antioxidant potential, lipid peroxidation, mitochondrial potential membrane and apoptotic induction by SF extracts on proximal and Cell distal tubule viability epithelial was cells. determined using the determined using MTT a assay. flow cytometric Mitochondrial JC-1 Mitoscreen membrane Caspase-Glo assay.and assay Glutathione GSH-Glo™ potential Cellularthe using measured were glutathione was and apoptosis The IC mg/mL, respectively. SF extracts significantly decreased( intracellular glutathione LLC-PK in PK mitochondrial membrane depolarization in ( both LLC-PK (2.2-fold; PCT and DCT epithelia have different cell architectures. the enzymes glutamate decarboxylase, GABA transaminase and succinic semialdehyde dehydrogenase, semialdehyde succinic and transaminase GABA decarboxylase, glutamate enzymes the and is transformed into citric acid cycle intermediates. proximity of filtered substances to this kind of tubular epithelium increases the susceptibility of these cells to damage. potential (hydroxyl free radical and superoxide neutrophil systems. scavenging properties) in cell-free and inhibition of HIV-1 target enzymes, such as HIV-1 reverse transcriptase. stimulated The route of elimination of polar compounds from kidney the functions to filter blood, allowing circulation substances to enter Bowman’s capsule occurs and the renal tubules. via the renal system. The Filtered nutrients are actively reabsorbed at the actively reabsorbed proximal at the convoluted distal convoluted tubule tubule (DCT). (PCT), while ions are The PCT epithelium has a brush border of tall microvilli that extends surface into area the 20-fold lumen for to the increase efficientthe reabsorption of molecules from the glomerularcirculation. Histologically, filtratePCT backcells stain into intensely with eosin due to their high content of organelles and mitochondria. The PCT is responsible for the active acids reabsorption from of the glomerular 99% filtrate. of glucose and amino less intensely due to fewer organelles) that actively reabsorb sodium from the tubular fluid. tRNA synthetase, and consequently can be incorporated into newly synthesised peptides. produced by arginase-mediated hydrolytic cleavage of L-canavanine, has been tumour properties. shown to have anti- patients. the treatment of wasting in cancer and HIV/AIDS GABA, an inhibitory neurotransmitter, mediates most of its effects inside the nervous system. been used as a drug for the relief of anxiety and stress. the absorption of ions in the renal tubules. Many experiments have been done to determine the mechanism(s) of vivo action experiment it of was SF concluded that extracts. SF In extracts possessed an anticonvulsant in effects in mice subjected to the induction of epilepsy. morphological characteristics of apoptosis and cell growth inhibition. SF was demonstrated to be concentration-dependent in breast cancer and leukaemia cell lines, with no effects. antioxidant significant Sutherlandia frutescens endemic to South Africa. by traditional healers to treat a variety of ailments including internal cancers, diabetes, uterine disease, influenza, HIV, depression, and arthritis. effects. humans, but have produced no known side Leaves of saponins, parabens, (GABA), acid stores nitrogen in seeds and SF is is dependent used on the in availability plant of abiotic structural chemical analogue of factors. L-arginine. It defense can be recognised by mechanisms. arginine-utilising enzymes such as arginyl- utherlandia S
of proximal
1 ; 1 1 Sutherlandia frutescens s effect
he Sutherlandia frutescens T Discipline of Medical Publishing. This work is licensed under the Creative Commons Attribution License. © 2010. The Authors. Licensee: OpenJournals This article is available at: http://www.sajs.co.za epithelial cells. S Afr J Sci. 2010;106(1/2), Art. #10, 5 pages. DOI:10.4102/sajs. v106i1/2.10 Chuturgoon AA. The effects of extracts in cultured renal proximal and distal tubule Published: 11 Mar. 2010 How to cite this article: Phulukdaree A, Moodley D, Dates: Received: 03 Sept. 2009 Accepted: 08 Dec. 2009 Sutherlandia frutescens antioxidant; lipid peroxidation; mitochondrial depolarisation; apoptosis of KwaZulu-Natal, Private Bag 7, Congella 4013, Durban, South Africa Keywords: Postal address: Discipline of Medical Biochemistry, School of Medical Sciences, University email: [email protected] Durban, South Africa Correspondence to: Anil Chuturgoon Biochemistry, School of Biochemistry, School of of Medical Sciences, Faculty Health Sciences, University of KwaZulu-Natal, Anil A. Chuturgoon Affiliation: 1 Authors: Alisa Phulukdaree Devapregasan Moodley http://www.sajs.co.za The effects of Research Letter Phulukdaree, Moodley & Chuturgoon
To date, limited scientific evidence has been available on the from the blanks was aliquoted into a microtitre plate. The optical mechanism by which SF extracts affect cellular processes and the density was measured at 532 nm, with a reference wavelength side effects related to their use. This medicinal plant, however, of 600 nm, by an ELISA plate reader. The sample means of continues to be recommended as a traditional remedy and is ten replicates were calculated and divided by the absorption used by a large portion of the South African community. In coefficient, 156 mM-1. this study, the nephrotoxic and apoptotic effects of SF extracts on two kidney cell lines, LLC-PK (PCT epithelium) and MDBK 1 Glutathione assay (DCT epithelium), were investigated and compared. The GSH-GloTM Glutathione Assay (Promega, Madison, USA) was used to measure glutathione (GSH) levels. Cells (those MATERIALS AND METHODS treated with SF extract and the untreated controls after 48 h Materials incubation) were transferred to an opaque microtitre plate (50 Sutherlandia frutescens tablets (Phyto NovaTM, Cape Town, South µL of 10 000 cells/well, 10 replicates). GSH standards (0 µM – 5 µM) were prepared from a 5 mM stock solution diluted in Africa) were purchased from a local pharmacy. The LLC-PK1 and MDBK cell lines were purchased from Highveld Biologicals water. Five two-fold dilutions of the GSH stock were prepared (Johannesburg, South Africa). All tissue culture reagents, the and transferred into wells (50 µL) of the microtitre plate. GSH-GloTM Glutathione Assay and the Caspase-Glo® 3/7 Assay The 2X GSH-GloTM Reagent was prepared according to the were obtained from Whitehead Scientific (Johannesburg, South manufacturer’s instructions, added to the experimental wells Africa). The JC-1 dye was purchased from BD Biosciences (South (50 µL/well), and incubated at room temperature. Reconstituted Africa). All other reagents were purchased from Merck (South Luciferin Detection Reagent (50 µL) was added to each well and Africa) unless otherwise stated. incubated. The luminescence was measured on a ModulusTM microplate luminometer (Turner Biosystems, Sunnyvale, USA). Preparation of Sutherlandia frutescens extracts A standard curve was derived using the GSH standards (0 µM – 5 µM) and the GSH concentration in each sample was TM Phyto Nova Sutherlandia tablets were used to prepare an extrapolated from the equation. aqueous extract of the active ingredients of the plant. Sixty tablets were crushed to a fine powder in a pestle and mortar, weighed and suspended in deionised water (1.2 g per 10 mL). Caspase-3/7 assay The mixture was continuously stirred at room temperature for The apoptotic potential of SF extracts on both cell lines was 1.5 h, and thereafter transferred to 50 mL sterilin tubes and determined using the Caspase-Glo®3/7 assay (Promega). centrifuged (3 645 g, 10 min) at room temperature. The upper Caspase-Glo®3/7 Reagent was reconstituted according to the aqueous layer (SF extract) was removed, vacuum filtered and manufacturer’s instructions and added to both the SF-treated stored at 4 °C. SF extract dilutions (24 mg/mL, 12 mg/mL, 6 and control cells (following 48 h incubation) in the wells of a mg/mL, 2.4 mg/mL, 1.2 mg/mL, 0.6 mg/mL, and 0.3 mg/mL) microtitre plate (10 µL reagent per 50 µL of 10 000 cells/well, 10 were prepared using complete culture media (CCM), comprising replicates) and incubated in the dark (30 min). The luminescence Eagle’s minimum essential medium, 10% foetal calf serum, 1% was measured on a ModulusTM microplate luminometer (Turner L-glutamine and 1% penstrepfungizone. BioSystems). The caspase-3/7 activity of the SF-treated samples was represented as X-fold change compared to the control (cells Cell culture and cytotoxicity assay incubated with CCM only). LLC-PK and MDBK cells were cultured (37 °C, 5% CO ) to
Article #10 1 2 confluency in 75 cm3 flasks in CCM. The cytotoxicity of SFin Mitochondrial membrane potential 22 The mitochondrial membrane potential (∆Ψ ) of LLC-PK LLC-PK1 and MDBK cells was measured using the MTT assay. m 1 and MDBK cells (both those treated with SF extract and their LLC-PK1 and MDBK cells (10 000/well) were incubated with varying SF extract dilutions for 48 h in triplicate in microtitre untreated controls; 48 h) was assessed using fluorescence- activated cell sorting (FACS) and the JC-1 Mitoscreen assay South African Journal of Science South plates, together with the respective controls (cells incubated with CCM only). The cells were then incubated with the MTT (BD Biosciences) according to the manufacturer’s instructions. substrate (5 mg/mL) for 4 h. Thereafter all supernatants were Cells (approximately 100 000) were transferred to polystyrene aspirated, and dimethyl sulphoxide (DMSO) (100 µL/well) was cytometry tubes. The JC-1 dye (150 µL) was added to the cells
added to the wells. Finally, the optical density was measured at and allowed to incubate (37 °C, 5% CO2, 10 min). Cells were 570 nm, with a reference wavelength of 690 nm, by an enzyme- then washed with JC-1 Mitoscreen wash buffer (400 g, 5 min) linked immunosorbent assay (ELISA) plate reader (Bio-Tek and resuspended in 300 µL flow cytometry sheath fluid. Flow µQuant). The data were translated to ‘percentage cell viability’ cytometry data from stained cells (15 000 events) was obtained using a FACSCalibur (BD Biosciences) flow cytometer with versus ‘concentration of extract’, from which the IC50 (half the maximal inhibitory concentration) values for each cell line and CellQuest PRO v4.02 software (BD Biosciences). Cells were for the combination of treatments were determined. For all gated to exclude debris using FlowJo v7.1 software (Tree Star subsequent biochemical assays, both cell lines were grown to Inc., Ashland, USA)
confluency and treated with the determined IC50 values of the SF extracts. Statistical analysis Results are expressed as the means, with error bars representing Lipid peroxidation assay the standard deviations (s.d.) of the means. Statistical significance Oxidative damage of both cell lines was assessed using the thiobarbituric acid assay, because lipid peroxidation is TABLE 1 Viability of PCT and DCT cells incubated with SF for 48 h using the MTT assay commonly quantified by levels of malondialdehyde (MDA).
After the 48 h incubation with SF extract, the culture fluid from SF (mg/mL) LLC-PK1 CELL VIABILITY (%) MDBK CELL VIABILITY (%) each SF-treated flask and from the untreated controls (500 µL) was dispensed into duplicate glass tubes (one representing 24 25 ± 0.0* 15 ± 0.3* the sample, one representing a negative control), followed by 12 72 ± 2.1 24 ± 14.0*
addition of 7% H2PO3 (200 µL). A positive control of 1% MDA 6 89 ± 33.0 103 ± 12.7 was prepared. Thiobarbituric acid (1%, w/v)/ 0.1 mM butylated hydroxytoluene solution (400 µL) was added to sample tubes. 2.4 107 ± 10.9 118 ± 3.7 To the tubes with the negative controls, 400 µL of 3 mM HCl 1.2 103 ± 2.9 122 ± 9.3 was added. The solution was adjusted to pH 1.5 and heated at 0.6 122 ± 14.0 96 ± 7.9 100 ○C for 15 min. Once cooled, butanol (1.5 mL) was added 0.3 133 ± 11.0 101 ± 10.3 and the sample then centrifuged (8 400 g, 6 min). Following centrifugation, the butanol phase (200 µL) from each sample and *p < 0.001
55 S Afr J Sci Vol. 106 No. 1/2 Page 2 of 5 http://www.sajs.co.za Article #10 56 South African Journal of Science
. p < , at p and Figure were 22 S Afr J Sci S in vitro Research Letter Research < 0.0001) over p s an abundance
< 0.0001) of MDA in p cells had a significantly significantly a had cells 1 and MDBK cells 1 and MDBK cells, are affected 1 LLC-PK caspase 3/7 in both SF-treated is a good indicator of apoptosis m LLC-PK Ψ (as a fold-change in comparison with . This sensitivity may be attributed to DISCUSSION Cells treated with SF extracts
20 20 The SF-treated The in vitro . measured m Ψ ∆ < 0.0001) percentage of depolarised mitochondria as cells showed a highly significant 11.9-fold increase ( 1 p The crude plant concoction is normally main taken route orally and of the elimination of compounds is the via the renal system. absorbed The potential for constituent injury by polar noxious compounds in the PCT region is high due to the ability filtered been have that substances concentrate to cells PCT the of by the glomerulus. The results of this investigation have of shown renal that tubular both cells, kinds LLC-PK by SF extracts the location and functional capacities of these cells. The intact PCT is susceptible to injury, as it is at this site where of the toxicants nephron accumulate and where there i of mitochondria for active re-absorption and transportation of ions, low molecular weight and proteins, heavy glutathione metals. conjugates concentrations of 6 mg/mL and lower, displayed greater than 89% viability. At such low concentrations, SF extracts appeared to promote cell metabolism conversion of the as MTT salt. This evidenced might have been due by to both increased the increased mitochondrial increased reductase availability of reducing equivalents enzyme such as NADH, activity, levels of lipid oxidation products in both SF-treated cell lines. There were significantly higher levels ( both the SF-treated cell lines as compared with the 2B). (see Figures 2A and control cells respective potential analyses Mitochondrial membrane To determine whether higher ( compared with the untreated cells (80.2% vs 54.6%) (see 3). Similarly, the MDBK cells had a significantly higher ( The intracellular activities of cell lines were the relevant controls). The activity of caspase 3/7 in SF-treated LLC-PK < 0.0001) compared with the controls. cells also showed a significant 2.2-fold increase ( The SF-treated MDBK were cells PCT that indicated results These 2). Table (see controls more susceptible to apoptotic induction by SF than were DCT cells. SF is used by many South or Africans ameliorant as a for traditional remedy many diseases, including HIV infection. metabolically viable after SF-extract treatment, we investigated in changes 0.0001) percentage of depolarised mitochondria as 58.2%) (see Figure 3). with controls (81.7% vs compared Caspase-3/7 assay A significant change in ∆ FIGURE 2 < p Levels of MDA in SF-treated cells Levels of MDA < 0.001) < 0.0001) [B] treated with SF were significantly higher than in their respective controls. Results are presented as the mean mean the as presented are Results controls. respective their in than higher significantly were SF with treated [B] 0.0001) < p p < 0.0001). < p < 0.001) of *** p Vol. 106 No. 1/2 Page 3 of 5 Vol. for SF extract 50 cells as compared 1 cells treated with SF extracts ( extracts SF with treated cells 1 extracts
< 0.0001) [A] and MDBK cells ( cells MDBK and [A] 0.0001) < p and MDBK were different from each other and MDBK were different 1 and to 15% and 24% ( , FIGURE 1 cells ( cells 1 RESULTS cells 1 and MDBK cells treated with a range of 1 < 0.0001), whilst there also was a significant p < 0.0001). p *** Sutherlandia frutescens < 0.0001) (see Figure 1). Figure (see 0.0001) < < 0.0001). Results are presented as the mean ± s.d. and n = 10. ( 10. = n and s.d. ± mean the as presented are Results 0.0001). < p p and MDBK cells, respectively. The cell viability of both of viability cell The respectively. cells, MDBK and 1 Basal GSH concentrations in LLC-PK Basal GSH concentrations in The concentrations of MDA in LLC-PK in MDA of concentrations The ± s.d. and n = 10. ( Lipid peroxidation assays In order to determine cytotoxicity were related whether to oxidative stress, mitochondrial we measured damage the and decreased significantly in SF-treated LLC-PK with controls ( decrease in GSH in SF-treated MDBK ( cells controls as compared with GSH assays The intracellular concentrations of treated GSH in renal both epithelial SF-extract- cell lines were determined. They viability was decreased to 25% and 72% of controls ( in the case of LLC-PK controls in the case of MDBK cells. LLC-PK cell lines treated with concentrations between 0.3 6 mg/mL was more mg/mL than 89%. However, and at higher SF extract concentrations of 24 mg/mL and 12 mg/mL, respectively, cell The cytotoxic effects of SF extracts (MTT assay) were determined determined were assay) (MTT extracts SF of effects cytotoxic The in both LLC-PK extract dilutions for 48 h (see Table 1). The was determined IC as 15 mg/mL and 7 mg/mL dilutions for the La Jolla, USA). Cytotoxicity The statistical significances of the lipid peroxidation assay, GSH assay depolarisation mitochondrial and assay caspase-3/7 assay, were determined by the Mann-Whitney test for non-parametric data using GraphPad InStat Software, (GraphPad Software Inc., between samples for the MTT assay was determined using the one-way ANOVA Tukey Kramer Multiple Comparisons test. 0.0001) compared with the relevant controls. The MDBK cells treated with SF also showed a decrease decrease a showed also SF with treated cells MDBK The controls. LLC-PK in significantly decreased were GSH of levels The relevant the with compared 0.0001) ( levels GSH in http://www.sajs.co.za The effects of Research Letter Phulukdaree, Moodley & Chuturgoon
cells were better protected from increased ROS production and subsequent peroxidation than the MDBK cells were.
The oxidative ability of L-canavanine to induce ROS and ensuing lipid peroxidation was determined previously in a mouse glial cell line (N11)27. In this study, a luminol-amplified chemiluminescence assay showed that 1 mM of L-canavanine significantly p ( < 0.05) increased ROS production and lipid peroxidation in N11 cells.27 L-canavanine, a structural analogue of L-arginine, is a competitive inhibitor of arginine-utilising enzymes such as nitric oxide (NO) synthase, which synthesises NO from L-arginine.28 Nitric oxide is involved in many biological processes and has the ability to spontaneously react with superoxide radicals to produce peroxynitrite and subsequently, nitrate.27 Nitric oxide, therefore, serves as a reactive radical Both cell lines contained approximately 50% depolarised mitochondria without SF treatment. scavenger that ultimately prevents oxidative damage to cellular Results are presented as the mean ± s.d. and n = 15 000. (***p < 0.0001). components.29 The possible decreased activity of NO synthase FIGURE 3 Depolarised mitochondria determined by the JC-1 Mitoscreen assay of control and and inhibition of NO synthesis by SF may explain the significant lipid peroxidation in these renal cell lines. SF-treated LLC-PK1 and MDBK cells Another possibility for the increased lipid peroxidation in
TABLE 2 both the SF-treated LLC-PK1 and MDBK cells may be due to a Caspase-3/7 activity of PCT and DCT cells incubated with SF for 48 h. decreased ∆Ψm. GABA was shown to penetrate up to 60% of RLU – relative light units the mitochondrial matrix volume in rat brain and liver cells.
p The weak acidic nature of GABA may contribute to the proton Control – Caspase-3/7 SF – Caspase-3/7 Fold 30 activity (RLU) activity (RLU) Change value gradient disruption within mitochondria. A change in ∆Ψm promotes formation of ROS as it offsets the normal functioning p < of the ETC. LLC-PK1 16 127 ± 346 191 706 ± 13455*** 11.9 0.0001 p < In addition to its acidic nature, high concentrations of GABA MDBK 5 137 ± 576 11 365 ± 486*** 2.2 0.0001 cause the reduction of intra-mitochondrial NAD+ stimulated by ***p < 0.0001 glutamate.30 Excessive intra-mitochondrial NADH is dissipated by donating its electrons to the ETC. The metabolism of GABA a byproduct of metabolic pathways such as glycolysis and the involves the enzymes GABA: pyruvate transaminase, GABA: Krebs cycle.23 The anti-diabetic and favourable cellular metabolic α-ketogluterate transaminase and succinic semialdehyde properties of SF were previously demonstrated in male Wistar dehydrogenase to form succinate which can enter the Krebs 14 rats.24 cycle and produce substrates for the ETC.
The hypoglycaemic potential of SF was shown in diabetogenic ROS production in the mitochondria increases susceptibility rats, where it exhibited properties similar to those of the extracts for membrane damage, altering the mitochondrial permeability
Article #10 similar to those of metformin, the common diabetes type-2 transition. This may account for the high percentage of drug.24 SF extracts increased the uptake of glucose by the kidney depolarised mitochondria noted after treatment of cells with SF extracts. The alteration of the ∆ within a cell will not, however, and resulted in increased glycolysis, which increased the rate Ψm 31 of the Krebs cycle and produced more reducing equivalents occur simultaneously to all the mitochondria that are present. The number of mitochondria that are affected determines the (NADH and FADH2). The high concentration of reducing South African Journal of Science South equivalents donate electrons to the electron transport chain amount of ATP generated and the ultimate fate of the cell. The (ETC), resulting in increased ATP production via oxidative amount of ATP available will determine the type of cell death 20 phosphorylation. This increased ATP concentration, is utilised that occurs within the cell. for the active reabsorption of substances by the PCT and DCT. The continuous flux of electrons along the ETC and pumping When mitochondrial membranes are depolarised, pro-apoptotic signals are released. It was demonstrated that changes in ∆Ψ of protons out of the matrix results in a negative mitochondrial m membrane potential, during which process reactive oxygen favoured the transition of a tightly bound form of cytochrome species (ROS) are generated as a byproduct, rendering the c (to cardiolipin in the inner mitochondrial membrane) into mitochondria susceptible to oxidative damage.25 its loosely bound form, followed by the release of cytochrome c into the extra-mitochondrial environment.32 The release Free radicals attack polyunsaturated fatty acids, a major of cytochrome c, Apaf-1 and calcium ions into the cytosol results in the activation of downstream caspases that execute component of cell membranes, thereby forming lipid peroxyl 25,33,34 radicals. Lipid peroxidation is initiated when the reactive lipid apoptosis. This study showed that SF-extract treatments peroxyl radicals are not detoxified by antioxidants. The lipid increased caspase-3/7 activity in both cell lines (by 11.9-fold in LLC-PK cells and by 2.2-fold in MDBK cells). alkoxyl radical undergoes cyclisation forming an intermediate 1 product which can degrade into MDA.26 Lipid peroxides can be detoxified by conjugation to antioxidant molecules such as When comparing the change in ∆Ψm of LLC-PK1 and MDBK ascorbic acid, α-tocopherol, uric acid and GSH.27 Glutathione cells with caspase-3/7 activity, it became evident that SF extracts concentrations were significantly decreased in SF-treated LLC- are potent apoptotic inducers. The percentage mitochondrial membrane depolarisation in both cell lines appears to be equal PK1 and MDBK cells with a corresponding increase in lipid peroxidation compared to the controls (p < 0.0001). at basal levels; following SF-extract treatments, an almost similar
increase in depolarisation occurs in both LLC-PK1 and MDBK cells. Both the LLC-PK1 and MDBK cells were transformed cell lines which proliferated rapidly and possessed high metabolic activity
which resulted in increased production of ROS, which, when The 11.9-fold increase of caspase activity in LLC-PK1, as not sequestered rapidly, increased lipid peroxidation. The MDA compared to a 2.2-fold increase in MDBK cells, may be due to the
concentrations in the control LLC-PK1 and MDBK cells were 0.14 difference in mitochondrial numbers and cellular architecture mM and 3.0 mM, respectively (see Figures 2A and 2B), whereas of both cell lines. It is known that PCT epithelium contains
the concentrations of GSH in LLC-PK1 and MDBK cells were 2.85 more mitochondria than do DCT cells. The depolarisation µM and 2.1 µM. respectively (see Figure 1). The increased basal of mitochondria allows for the increased release of more pro-
GSH concentration in the LLC-PK1 cells suggested that these apoptotic signals, thereby resulting in increased apoptosis.
57 S Afr J Sci Vol. 106 No. 1/2 Page 4 of 5 http://www.sajs.co.za Article #10 58 South African Journal of Science
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